Fission in a colonial marine invertebrate signifies unique life history strategies rather than being a demographic trait

Each of the few known life-history strategies (e.g., r/K and parity [semelparity and iteroparity]), is a composite stratagem, signified by co-evolved sets of trade-offs with stochastically distributed variations that do not form novel structured strategies. Tracking the demographic traits of 81 Botryllus schlosseri (a marine urochordate) colonies, from birth to death, we revealed three co-existing novel life-history strategies in this long-standing laboratory-bred population, all are bracketed through colonial fission (termed NF, FA and FB for no fission, fission after and fission before reaching maximal colony size, respectively) and derived from organisms maintained in a benign, highly invariable environment. This environment allows us to capture the strategists’ blueprints and their net performance through 13 traits, each branded by high within-strategy variation. Yet, six traits differed significantly among the strategies and, in two, the FB was notably different. These results frame fissions in colonial organisms not as demographic traits, but as pivotal agents for life-history strategies.

Reproductive statuses (RS) scores. In developing B. schlosseri colonies, male gonads appear first, and two to four blastogenic cycles later, they were followed by the development of female gonads. Mature colonies may go through short or long periods of sexual sterility or may develop male gonads only 37,38 . Further, reproductive statuses (hermaphroditism, male-only, sexual sterility) may vary between different colonies under the same environmental conditions 37 . For the RS score, sexual sterility was graded as 0, male-only as 1 and hermaphroditism as 2. RS scores did not consider the average number of female/male gonads per zooid/bud. Observations were taken once every 15 days, such that the onset of reproductive activity in colonies could only be missed by a single blastogenic cycle (duration: 1 week at 20 °C). Following fission, an average RS score was calculated for the whole genet. In addition, we documented qualitatively the reproductive efforts' outbreaks (RO). While we did not count the number of gonads/embryos as part of the observations, unusually high and fast changes in the number of ova in buds or embryos in zooids were observed and documented. Along the observations, we recorded extreme events where high numbers of embryos and/or ova per zooid were documented and these events were noted as "reproductive effort's outbreak" to highlight qualitatively these episodes.
To delineate either a single or repeated sexual-reproduction cycle in colonies to reveal repeated male-only and hermaphroditic states, we defined each reproductive cycle as from the male-only state to the end of the subsequent hermaphroditic state, each termed as a RS segment (RSS).

Colony fission.
A fission event is characterized by a gradual splitting process, developing in the tunic matrix between colonial systems, following which the colonial entities, reaching a minimum size of two systems, were physically split into two or more disconnected ramets. Colony fission 37 (Fig. 1d-h) may take days to weeks from onset to completion, and was determined to have begun with the first observation in which subclones of colonies were not connected by a single blood vessel. The number of zooids following fissions events revealed the sum of zooids in all ramets for any specific colony (genet). The colonial entity is composed of zooids, clustered here in a single flower-like shape named "system", containing six zooids (two zooids are schematically drawn, depicting internal organs). Three asexually derived generations are seen in the colony, the functioning zooids, the primary buds (pb), and the secondary buds (sb). Male gonads and female gonads are located in the zooids and buds, while embryos are found only in zooids. bs = branchial sac, em = embryo, en = endostyle, oo = oocyte, pb = primary bud, sb = secondary bud, si = siphon, ts = testes, tu = tunic, zo = zooid. (b) A colony is developed through blastogenesis, repeated cycles of life and death, each including four stages (A to D; sensu 62,63 ). Each colony consists of three consecutive generations of modules at different developmental stages. Every blastogenic cycle lasts about 1 week at 20 °C and concludes in the death and absorption of the oldest generation, the zooids, while the younger generations, the buds and budlets, are developing. (c) A single Botryllus colony that underwent several fission events and is currently divided into 14 ramets. Ramets exhibit different CV scores (encircled numbers, see Suppl. Figure 1). The mean CV score of the colony (the sum of all ramets, the genet) is 2, taking into consideration the number of zooids in each ramet (d-g). Colony FB19 undergoing two fission events over four sequential observations. Age and genet size are mentioned in the lower-left part of each figure (zo = number of zooids). (d) Day 225: the colony can be seen as a single entity (e, e′). Day 240: the lower extension of the colony is retreat-growing (sensu 39 ) from the upper part of the colony and both are still connected by a single blood vessel. (f) Day 255: the colony is split into two colonial ramets. (g) Day 270: a second fission event occurs in the upper ramet, resulting in three ramets. (h) A fissioned colony may demonstrate discrete blastogenic stages in the separate ramets, hypothetically enabling it to self-breed. The upper ramet is in blastogenesis stage C, the left ramet is in early blastogenic stage D and the right ramet is in blastogenic stage A. Scale bars = 1 mm. www.nature.com/scientificreports/  www.nature.com/scientificreports/ Statistical analyses. All analyses used SPSS software. A one-way ANOVA followed by a Bonferroni Multiple Sample Test were employed on the following characteristics: life span, age at the onset of male/female gonads, total zooid numbers, age at the peak of the colony's size, maximum colony size and RSS length (days). A one-sample t-test was used to determine the age at the first fission; a Mann-Whitney U test on the number of fissions throughout life; the non-parametric Kruskal-Wallis one-way ANOVA test employed on the number of RSS segments and observations with male-only/hermaphroditic states; Chi Square tests on the outcomes of fission events in the two-fissioned life histories; and discriminant analyses to predict the clusters of the three life histories.; Pearson tests determined correlations between life span and the number of all RSSs and between life span and the number of fissions; and Repeated Measures ANOVA was used on the gross size of colonies before and after the first fission.

Results
General. We followed 81 colonies growing on glass substrates from birth to death for up to 3 years (average life span was 290 ± 157 days, Table 1; Supp. Tables 1, 2, 3) under relaxed laboratory conditions. The onset of reproduction (male gonads) appeared within the first 2 months of age (55 ± 20 days; n = 65; Table 1) and oocytes first appeared about a month later (80 ± 51 days; n = 75; Table 1). During their life spans, more than half of the colonies (n = 46; 57%) went through at least one fission event, including genotypes that showed repeated cycles of fast growth, degeneration (sensu 23,39 ), rejuvenation (sensu 40 ) and colony fissions. In the other 35 colonies, no single fission event was recorded throughout their life span. In the wake of the fission events, we defined three distinct life-history categories: (i) NF: colonies that did not experience a fission event throughout their entire lives; (ii) Fission type A (FA): colonial fission developed after the colony reached its peak number of zooids/observation; (iii) Fission type B (FB): colonial fission developed before the colony reached its peak number of zooids/observation. www.nature.com/scientificreports/ Fission as a demographic trait. The first signs of fission were witnessed when the typical colonial pattern of packed systems was interrupted, and each new developing colonial system in subsequent blastogenic cycles was spaced in the tunic matrix, consistently dispersed from neighbor systems (Fig. 1d-e′). Simultaneously, the semitransparent tunic turned opaque, developing imperfections in the fission areas (Fig. 1e). Each colonial-fission process was completed within several days/weeks from its onset through a gradual tunic deterioration, occurring simultaneously with the narrowing of connecting vasculature and the reduction of blood-cell circulation, followed by the decay of the tunic and connecting blood vessels, until the actual disconnection between parting ramets (Fig. 1f-g). Within colonies that underwent fission, the first fission occurred at the age of 168 ± 79 days (n = 46) in colonies experiencing on average 2.6 ± 2.1 fission events throughout their life, and up to nine times per genotype (Table 1) www.nature.com/scientificreports/ three life-history strategies (n = 35 for NF, n = 23 for FA, and n = 23 for FB colonies; Table 1, Fig. 2a- Tables 2, 3). NF and FA significantly differed from FB at the age of the onset of male and female gonads (Table 1). Reproductive outbreaks (RO) were also significantly disparate between NF, FA and FB colonies (Kruskal-Wallis; p = 0.021; Table 1). A Mann-Whitney U Post hoc revealed a significant difference between NF and FB (p = 0.008). A typical NF pattern shows a single peak in the number of zooids around midlife, followed by a gradual, constant decline, to a minimal colonial size before death, as well as a single RSS, composed of a male-only phase, followed by a 2-month-long hermaphroditic status, spanning the time of the colony's peak size (Fig. 2a,d). The fissioned FA and FB strategies are epitomized by repeated RSSs of high and low CV scores that repeatedly decline at the end of each RSS and raise at the beginning of a new segment, when colonies re-juvenilize, as is manifested by an upsurge in the number of zooids (data not shown). RSSs contained several male-only observations that were inserted between hermaphroditic states (FA: Fig. 2b,e; FB: Fig. 2c,f). Sexual-sterility phases (excluding the pre-reproductive stage of young colonies) were rare, recorded in 2 NF, 1 FA and 4 FB colonies, all at advanced ages, and which were accompanied by a significant zooid reduction. Further, fission was commonly followed by the loss of developmental synchronicity between ramets of the same genet (e.g., Fig. 1h).
A discriminant analysis of 65 colonies with complete data, which comprised the variables length of life span, age at the peak colony size, maximum size of the colony, ages of the onset of male gonads and of female gonads (Suppl.  Fig. 3a). Fig. 2d-f) was found in 74% of the NF colonies, 50% in the FA colonies and 9% in the FB colonies. The average RSS length (89 ± 45 days for 142 RS segments in 62 colonies) did not significantly differ between the three lifehistory strategies, even though the mean RSS in FB was 31% longer than in NF (Table 1, Suppl. Table 4).

Characterization of the RS segments (RSSs). A single RSS (delineated by vertical black lines in
By contrast, the number of RSSs significantly differed between the three life-history strategies ( Table 1, Suppl. Table 1). The Pearson correlation coefficient (r) for life span compared to the number of all RSSs was 0.8 (n = 77, p < 0.001), demonstrating a strong positive relationship between the two variables. Yet, examining proportions of male-only and hermaphroditic observations within a segment (Table 1; 57 colonies with 106 RSSs) revealed 35 ± 20% and 65 ± 20%, respectively, with no significant difference between the three life-history strategies ( Table 1), suggesting that reproductive statuses within segments are not a variable trait when considering the colonial strategic life span.

Fissions shape astogeny. A Pearson correlation between life span and the number of fissions in FB colo-
nies revealed a correlation coefficient (r) of 0.68 in FB (p < 0.001) compared to 0.33 for FA (p = 0.1). Yet, the time of the first fission (a baseline fission, as additional fissions did not develop in all ramets) in all fissioned types (n = 46) was 168 ± 79 days (Table 1), with no significant differences between the FA and FB strategies (Table 1), suggesting an inherent fission trait in astogeny. Focusing on the first fission event, in 96% of FA colonies, fission was followed by an immediate (the first observation following fission) decrease in colonial size, whereas 62% of fissions in FB colonies resulted in an immediate increase in colonial size ( Fig. 3c; chi-test, chi square = 16.8, df = 1, p < 0.001), an outcome further supported by analyses performed on the first and second fission events ( Fig. 3d; F = 4.4; df = 1.8, 12.8; p = 0.019). While total sizes of FA colonies declined after fission (before: 235 ± 155 zooids; after: 99 ± 88 zooids), in FB colonies, total sizes increased (before: 146 ± 78 zooids; after: 616 ± 626 zooids). A Repeated Measures ANOVA test on the changes in the means, revealed no significant difference between the two time-points in the two fission types (F = 0.369, df = 1, p = 0.547).

Discussion
All life-history strategies have been marked by co-evolved sets of trade-offs, which are the constrained lineages between traits, revealing an 'optimization' of trade-offs for growth, survival and reproduction [41][42][43] . Clearly, an optimal life-history strategy may be different for each species, depending on its genetic background, past and present environment, and other biological and environmental interactions and constraints. Yet, these life-history strategies illustrate the flexibility of within-species life histories, which allow organisms to endure various conditions and survive strong stochastic-disturbance events [44][45][46] . Flexible life-history characteristics among individuals of a specific taxon can further influence the magnitude of demographic stochastics, maximizing the extent to which offspring of parents with distinct life-history strategies are differentially staged at the population level, and further conferring diverse fitness constraints [46][47][48] .
The interplay of survival, life span, growth rate and reproductive status define life-history strategies across taxa. Each such life-history strategy is a composite stratagem and banks on the coordination of several www.nature.com/scientificreports/ co-functionating traits. While the literature attests for a high diversity of demographic properties and traits within populations and large amounts of phenotypic variation and trade-offs among individuals, with regards to size, survival, onset of reproduction and total reproductive outputs 41,45,[48][49][50] , the number of life-history strategies remains minimal, as the different variations and constraints revealed by different taxa distribute stochasticity, without forming novel structured strategies along a continuum (such as r and K strategies, or parity, semelparity and iteroparity 51,52 . The reiteration of modules and the ability of colonial organisms to create independent ramets provide colonies with the proficiency to develop, like weeds, bushes, or trees, bodily structures that solitary animals cannot 53 . While individuals of unitary organisms appear to hold different positions along the r and K strategy continuum of life-history strategies 43,46,47,50,54 , with the employment of fission, fusion and partial mortality, colonial organisms may "deceive" with regards to the correlations between age/size/reproduction 1,3,4,55 and thus rebuff the accepted notion of structured life-history traits for large vs. small phenotypes in colonial organisms. Three alternative life-history strategies (NF, FA, FB; Fig. 4) which reflect genetically based ecological consequences, co-exist in a long-term laboratory-bred B. schlosseri population. All are categorized through the phenomenon of colonial fission (no fission, fission after/before reaching maximal colony size) and derived from organisms maintained under benign, further assumed to be highly invariable, environmental (laboratory) conditions. NF, FA and FB strategies thus underlie the core expression of the traits' phenotypes. Moreover, the benign environment allows for the capture of the genetic blueprints of these strategies and their net performance (e.g., fission has been developed without any recognized biological or environmental driver, contrasting with existing literature; see 56 ; Herrera-Cubilla et al., 57 ; Bingham et al., 58 ). The Botryllus NF type is short-lived and reaches maximum size and sexual maturity at an early age. The NF type also has the smallest accumulated soma size and the fewest RS segments over its life. By contrast, the FA type is a phenotype with a medium-length life span that reaches maximum size at a mid-age and sexual maturity at an early age. The FA type also has an intermediate accumulated soma size and a middling number of RS segments over their lives. Finally, the FB type is a longlived phenotype that reaches maximum size and sexual maturity later in life. The FB type also has the highest accumulated soma size and the most RS segments over their lives.
It has been advocated that functional ecological traits could be related to any species' demography 59 , an argument primarily evidenced in field-oriented studies (e.g. 60 ). Rather than defying demographic trade-offs, this study presents basic associations between fission and divergent life-history traits in B. schlosseri in a relaxed environment, configuring the framework for each of the three coexisting, starkly different (Fig. 4), yet variable, life-history strategies. Thirteen traits were followed, branded by high within-strategy variation, yet six traits (life span, total accumulated soma, age at peak size, maximum colony size, RS segments and number of fission events) differed significantly between the three strategies. In two traits (ages of onset of male/female gonads), the FB was notably different, and only four traits (RSS length, male-only state in segments, hermaphroditic state in segments and age at first fission) were not significantly different from each other (Fig. 4). The expression of high variability associated with any trait, as exemplified by the 81 genets that were followed here from birth to death, reveals a continuous range for each trait's metric (broached as phenotypic plasticity), together with a clear distinction between the three life-history strategies.
The main conclusion of the present study is that fission in colonial organisms should not be solely understood within the framework of a demographic trait per se 10,14,56-58 but should preferably be considered as a trait along a continuum (sensu 43,46 ), reflecting a major attribute of a life-history strategy. Observing the expression of fission under relaxed (benign) environmental conditions allowed us to identify colonial fission with the framework of distinct life-history strategies, in concert with a suite of other continuously varying traits (Fig. 4). Even with the expressed high within-trait variability, altogether the results revealed three finely graded but distinct life-history strategies. These intra-specific life-history strategies in B. schlosseri and their landscapes' variations, with aligned ratcheting fitness (a topic not studied here), are likely reflected in the successful invasiveness of this species, which has become cosmopolitan in temperate zones 30 www.nature.com/scientificreports/